System and method for high numeric aperture imaging systems

ABSTRACT

A system and method for high numeric aperture imaging systems includes a splitter, a defocusing system, and a combiner. The splitter reflects a portion of collected light and transmits another portion of the collected light. The defocusing system is configured to modify optical power of either the transmitted portion or reflected portion of the collected light. The combiner is oriented with respect to a mechanical angle. The combiner recombines portions of the transmitted portion and the reflected portion such that the transmitted portion and reflected portion are subsequently transmitted being separated by an optical separation angle based upon the mechanical angle of orientation of the combiner. Various other implementations are used to maintain focus with regards to the imaging systems involved.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 60/240,125, filed Oct. 12, 2000, incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to imaging systems, and moreparticularly to systems and methods for high numeric aperture imaginginvolving low-light and high-resolution, such as used for microscopicimaging of biological samples or macroscopic imaging of astronomicalsamples.

2. Description of the Related Art

Low-light, high-resolution imaging involves situations where relativelylittle amount of light reflects, scatters or emanates from a targetobject to be viewed by using a high-resolution imaging system. Suchlow-light, high-resolution imaging can involve microscopic imaging oftarget objects including biological samples prepared using fluorescencein situ hybridization (FISH) or can involve macroscopic imaging oftarget objects including stars.

Conventional imaging systems are overly challenged by low-light,high-resolution imaging. Objective components used in high-resolutionimaging systems need to have very high numeric aperture values.Unfortunately, a high numeric aperture value of the objective componentresults in a very small depth of field in which to view target objects.Small depth of field raises significant challenges in achieving andmaintaining focus of target objects to be viewed during low-light,high-resolution imaging. If focus of a target object is not achieved andmaintained, the resultant defocused image of the target object at adetector is spread over an unacceptably large area of the detector witha loss in spatial resolution and decrease in signal-to-noise ratioassociated with the image of the target object.

Some of the target objects involved with low-light, high-resolutionimaging further challenge focusing capabilities of conventionalhigh-resolution imaging systems. Study of these target objects requirescapture of three-dimensional aspects associated with the target objects.For instance, FISH probes, having as little as 10,000 fluorescingmolecules, are used to determine the presence of various chromosomes inbiological cells. The FISH probes are located in three-dimensional spacewithin the nucleus of the cell and can be oriented along the optic axisof the high-resolution imaging system. With the high sensitivity tofocus in conventional high-resolution imaging systems resulting from useof high numeric aperture objective components, light from a small pointsource, such as a FISH probe, is easily defocused and spread out over alarge area of an associated detector. If two FISH probes at the samelevel of defocus are imaged together, their detected images becomeindistinguishable from one another due to overlapping of the blurreddefocused FISH probe images.

Conventional attempts to remedy focus problems involved withhigh-resolution imaging with high numeric aperture objective componentshave been only partially successful. For instance, some high-resolutionimaging systems pan through the target object along the imaging systemoptic axis to acquire multiple image planes of the target object. Thesemultiple image planes are then analyzed to discriminate the presence ofmore than one target object, such as more than one FISH probe.Unfortunately, the significant amount of time required to collect andanalyze the multiple images, greatly limits the application of thisapproach. For example, this approach would not be readily applied tolow-light, high-resolution imaging of particles or cells moving in acontinuous flow past the high-resolution imaging system. Otherconventional attempts include simultaneously viewing a target objectfrom orthogonal directions, from opposite directions, or from viewsdefined by a strobe light. Shortcomings of these conventional attemptsinclude low throughput, poor resolution, lack of same plane objectdiscrimination, and object positioning difficulties.

Herein are described low-light, high-resolution imaging systems andmethods directed toward these and other issues. Other features andadvantages will become apparent from the following detailed description,taken in conjunction with the accompanying drawings.

SUMMARY OF THE INVENTION

A system and method for high numeric aperature imaging systems includesaspects directed to a first beam splitter configured to substantiallytransmit part of received light as first transmitted light and tosubstantially reflect part of received light as first reflected light.Further aspects include a defocus system configured to modify opticalpower of substantially one of the following: the first transmitted lightand the first reflected light, and to transmit the same as firsttransmitted defocused light. Additional aspects include a reflectorconfigured to reflect one of the following: the first reflected lightand the first transmitted defocused light. Further aspects include asecond beam splitter configured to substantially transmit part of one ofthe following: the first transmitted light as second transmitted lightand the first transmitted defocused light as second transmitteddefocused light and configured to substantially reflect part of one ofthe following: the first transmitted defocused light as second reflecteddefocused light and the first reflected light as second reflected light.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart illustrating a method for augmenting depth offield at a target object for low-light, high-resolution imaging.

FIGS. 2–3 are schematics illustrating an imaging system for low-light,high-resolution imaging.

FIG. 4–5 are schematics illustrating object planes associated with theimaging system, as shown in FIGS. 2–3.

FIG. 6–9 are schematics illustrating alternative implementations of theimaging system, as shown in FIGS. 2–3.

FIG. 10 is a schematic illustrating object planes associated with theimaging systems, as shown in FIGS. 6–9.

FIGS. 11 and 12 are schematics illustrating an alternativeimplementation of a component of the imaging system, as shown in FIGS.2–3 and 6–9.

FIGS. 13–14 are schematics illustrating an alternative implementation ofthe imaging system, as shown in FIGS. 2–3 and 6–9.

FIG. 15 is a schematic illustrating an alternative implementation of theimaging system, as shown in FIGS. 2–3, 6–9, and 13–14.

FIG. 16 is a schematic illustrating an exemplary set of images on adetector of the imaging system, as shown in FIGS. 2–3, 6–9, and 13–14.

FIGS. 17–18 are schematics illustrating an alternative implementation ofthe imaging system, as shown in FIGS. 2–3, 6–9, 13–15.

FIG. 19 is a schematic illustrating an exemplary set of images on adetector of the imaging system, as shown in FIGS. 17–18.

FIGS. 20–21 are schematics illustrating an alternative implementation ofthe imaging system, as shown in FIGS. 2–3, 6–9, 13–15, 17–18, and 20–21.

FIG. 22 is a schematic illustrating object planes associated with theimaging system, as shown in FIGS. 20–21.

FIG. 23 is a schematic illustrating a two-dimensional imaging systemusing active focusing.

FIG. 24 is a schematic illustrating an exemplary set of images projectedon one of the detectors of the two-dimensional imaging system, as shownin FIG. 23.

FIG. 25 is a schematic illustrating an alternative implementation of theimaging system, as shown in FIGS. 2–3, 6–9, 13–15, 17–18, and 20–21.

FIG. 26 is a flowchart illustrating a method used by alternativeimplementation of the imaging system, as shown in FIG. 25.

FIG. 27 is a schematic illustrating an alternative implementation of theimaging system, as shown in FIGS. 2–3, 6–9, 13–15, 17–18, 20–21 and 25.

FIG. 28 is a schematic illustrating an alternative implementation of theimaging system, as shown in FIGS. 2–3, 6–9, 13–15, 17–18, 20–21, 25, and28.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are systems and methods for achieving and maintainingfocus of target objects subject to low-light, high-resolution imaging.In general, light either reflecting, scattering or emanating from atarget object is collected and split into two or more light components.The optical power levels of some, but not all the light components arethen modified such that when the light components are recombined with anangular separation to form an image, each of the light components havedifferently positioned image planes where an object point of the targetobject is imaged.

For each image plane pair, a detector is such that each detectorreceives focused images from two object planes at the target objectassociated with the image plane pair of the detector to increase depthof field and focusing capability. In some implementations, focus isactively maintained though computer automated positioning of components.Other implementations actively maintain focus with a feedbackarrangement integral to a two-dimensional imaging system.

In the following description, numerous specific details are provided tounderstand embodiments of the invention. One skilled in the relevantart, however, will recognize that the invention can be practiced withoutone or more of these specific details, or with other equivalent elementsand components, etc. In other instances, well-known components andelements are not shown, or not described in detail, to avoid obscuringaspects of the invention or for brevity. In other instances, theinvention may still be practiced if steps of the various methodsdescribed could be combined, added to, removed, or rearranged.

A method 50 used by implementations of low-light, high-resolutionimaging systems is illustrated in FIG. 1. The method 50 first collectslight from a target object, such as emanating, scattered, reflected,and/or refracted light (step 58). The collected light is then collimatedby focusing to infinity (step 62). The collimated light is then splitinto a collective total consisting of two or more optical paths (steps66). Optical power is then added or subtracted from the light in chosenone or more, but not all, of the optical paths of the collective totalto defocus the light in the chosen one or more optical paths (step 70).Light in the collective total of optical paths is then recombinedwherein light of the one or more chosen optical paths has one or moresmall angular separations with respect to light of other optical pathsof the collective total (step 74).

The recombined light is then focused on one or more detectors resultingin one or more spatial separations of the imaged target object basedupon two or more image planes at the imaged target object associatedwith two or more object planes at the target object (step 78). Images ofthe imaged target object associated with the two or more image planesare then collected by the one or more detectors for analysis (step 82)and the method 50 ends to be ready for further imaging of other targetobjects. As an example, if two optical paths make up the collectivetotal, then optical power of only one of the paths is altered so thatthere is a spatial separation between two images resulting from the twolight paths on a detector. For an image plane defined by the detector,there are two conjugate object planes separated along the optical axisof the imaging system. Optical power of the one path is altered tocontrol the axial separation between the object planes so that the depthof field provided by the first image just overlaps the depth of fieldprovided by the second image to extend the total depth of field of theimaging system.

An implementation of an imaging system 100 as shown in FIG. 2 isconfigured to produce multi-focal plane images of a target object 102,such as biological cells or other small particles, being transported bya fluid flow, in the direction of the z-axis of FIG. 2, through a flowcell cuvette 104. The imaging system 100 has an unaltered optical path106 with a collection lens 108, an amplitude beam splitter 110 with abeam splitter optical coating 112, an amplitude beam splitter 114 with abeam splitter optical coating 116, an imaging lens 118, and a firstdetector 120 (shown in FIG. 3). The imaging system 100 also has adefocus optical path 122 with a first reflector 124, a defocus system126 being a negative lens 128 in the implementation shown, and a secondreflector 130 and sharing the collection lens 108, the beam splitteroptical coating 112, the amplitude beam splitter 114, the imaging lens118, and the first detector 120 with the unaltered optical path 106. Thetarget object 102, being the subject of imaging by the imaging system100 and found in the flow cell cuvette 104, emits, reflects, scatters,or refracts object light 132 to be received, collected, and passed bythe collection lens 108 as collected light 134 being collimated lighthaving generally parallel light rays being focused approximately atinfinity. The collected light 134 enters the amplitude beam splitter110, which splits the collected light into two optical paths having afirst transmitted light 136 and a first reflected light 138,respectively, in accordance with the beam splitter optical coating 112on the amplitude beam splitter.

The first transmitted light 136 is left unaltered and passes through theamplitude beam splitter 114 in accordance with the beam splitter opticalcoating 116 as second transmitted first transmitted light (2T1T light)138. The amplitude beam splitter 114 is oriented slightly by amechanical angle 104 with respect to the y-axis such that the secondreflected defocused light (2R defocused light) is oriented at an opticalangle of separation 142 with respect to both the x-axis and the 2T1Tlight 138. The 2T1T light 138 is then focused by the imaging lens 118 asimaged 2T1T light 144, which converges to focus at 2T1T image plane 146.The first reflected light 138 reflected by the beam splitter opticalcoating 112 is redirected by the first reflector 124 to pass through thedefocus system 126 thereby producing defocused first reflected light(defocused 1R light) 148, being decollimated light having optical powermodified by the defocus system. The defocused 1R light 148 is thenredirected by the second reflector 130 to pass through the amplitudebeam splitter 114 to be reflected in accordance with the beam splitteroptical coating 116. The 2R defocused light is brought to focus by theimaging lens 118 as imaged 2R defocused light 150, at 2R defocused imageplane 152.

The amount of defocus introduced by the defocus system 126 results inthe 2T1T image plane 146 and 2R defocused image plane 152 beingspatially separated from one another along the x-axis such that theirdepths of focus overlap. As shown in FIG. 3, the first detector 120 ispositioned with respect to a first detector image plane 154 and usesthis overlap of depths of focus of the 2T1T image plane 146 and the 2Rdefocused image plane 152 to effectively increase the overall depth offocus of the imaging system 100 for the first detector. The imagingsystem 100 provides, for the first detector 120 in the first detectorimage plane 154, two conjugate 2T1T object planes 156 and 2R defocusedobject plane 158 associated with an unaltered object light 160 and adefocus object light 162, respectively, as shown in FIGS. 3–5. As shownin FIG. 5, the 2T1T object plane 156 has a 2T1T object field depth 164and the 2R defocused object plane 158 has a 2R defocused object fielddepth 166, which have a first object field depth overlap 168. In otherimplementations the first object field depth overlap 168 may not existfor other tailoring of the depth of focus.

Implementations include the beam splitter optical coating 112 and thebeam splitter optical coating 116 being an amplitude beam splitter typewith transmittance and reflectance being nominally equal. The opticalcomponent of the amplitude beam splitter 110 and the amplitude beamsplitter 114 and their respective beam splitter optical coating 112 andbeam splitter optical coating 116 may have the coatings bonded betweentwo prism elements. Alternative implementations use plate or pellicleversions of the amplitude beam splitter 110 and the amplitude beamsplitter 114 with their respective beam splitter optical coating 112 andbeam splitter optical coating 116 being deposited on one surface. Insome implementations, the first reflector 124 and the second reflector130 are prisms, as illustrated, having total internal reflection fromuncoated surfaces. Other implementations of the first reflector 124 andthe second reflector 130 use reflective metallic or dielectric opticalcoatings deposited on surfaces including, but not limited to, a mirrorsurface of a plane mirror.

It is important to control intensities of the imaged 2T1T light 144 andthe imaged 2R defocused light 150, so that, typically, the imageintensities are substantially equal at the first detector 120. Intensitycontrol can be achieved in a number of ways. Depending upon the relativeoptical path efficiencies, such as the optical efficiency of theunaltered optical path 106 versus the optical efficiency of the defocusoptical path 122, it may be desirable to employ other than an equaltransmittance/reflectance ratio for the beam splitter optical coating112 or the beam splitter optical coating 116. For example, if theadditional optical elements in the defocus optical path 122 were toresult in more absorption loss relative to the unaltered optical path106, it would be beneficial to reflect more light at the beam splitteroptical coating 112 and transmit less light to the unaltered opticalpath to balance the light intensity in the imaged 2T1T light 144 and theimaged 2R defocused light 150. Commonly availabletransmittance/reflectance split ratios for commercially availablebeamsplitter coatings include 50/50, 60/40, 40/60, 30/70, and 70/30.Other implementations using other split ratios for light intensitycontrol are readily achievable with customized optical coatings known inthe art.

In addition to the choice of beamsplitter coating, such as choice of thebeam splitter optical coating 112 or the beam splitter optical coating116, light intensity can be controlled by placement of neutral density(ND) filters in the unaltered optical path 106 or the defocus opticalpath 122. In some implementations reflective or absorptive type filtersare used to reduce intensity in the unaltered optical path 106 or thedefocus optical path 122 to match that of the other. For instance, asingle filter of the appropriate density value is used in someimplementations to correct the mismatch while a variable density filtercomponent such as a stepped ND filter or linear wedge neutral densityfilter is used in other implementations where optical density of thecoating varies linearly with position as needed. Implementations using avariable density filter take advantage of its convenient light intensityadjustment and single design approach to compensate for variation incomponent efficiencies in a manufacturing environment.

In alternative implementations of the imaging system 100 illustrated inFIGS. 6–9, a second of the imaging lens 118 is used to focus thatportion of the first transmitted light 136 reflected by the beamsplitter optical coating 116 of the amplitude beam splitter 114 into animaged 2R1T light 170 on the 2R1T image plane 172. The second imaginglens 118 also focuses that portion of the defocused 1R light 148transmitted by the beam splitter optical coating 116 of the amplitudebeam splitter 114 into imaged 2T defocused light 174 onto the 2Tdefocused image plane 176. The 2R1T image plane 172 and the 2T defocusedimage plane 176 have a corresponding 2R1T object plane 178 and a 2Tdefocused image plane 180, respectively. The implementations also have asecond detector 182 along a second detector image plane 184 to receivethe 2R1T image plane 172 and the 2R defocused focus cell images 232. Oneimplementation also uses an additional reflector 186 to redirect the2R1T image plane 172 and the 2T defocused image plane 176. Asillustrated in FIG. 10, the 2R1T object plane 178 has a 2R1T objectfield depth 188 and the 2T defocused image plane 180 has a 2T defocusedobject field depth 190, which share a second object field depth overlap192. The 2R defocused object field depth 166 and the 2R1T object fielddepth 188 also share a third object field depth overlap 194.

As shown, the defocus system 126 can be implemented as the negative lens128. In other implementations, the defocus system 126 can be a positivelens element or a compound optical system configured to decollimateinputted collimated light. Implementations include lens elements beingground and polished or molded, being glass or plastic, being reflectiveor refractive, and having spherical or aspherical surfaces.Implementations using compound optical systems may include bothtransmissive and reflective optics. An exemplary compound optical systemimplementation of the defocus system 126 is illustrated in FIGS. 11 and12 where the first reflected light 138, as collimated light, enters afirst positive lens 194 and is brought to focus at an intermediate focalpoint 196. As shown in FIG. 11, a focused lens spacing 198 between theintermediate focal point 196 and a second positive lens 200 is set tothe focal length of the second positive lens such that a collimatedlight 202 leaves the defocus system 126.

The performance of the exemplary implementation illustrated in FIG. 11could be duplicated by any number of lens combinations conventionallyknown. In order to modify the optical power of the first reflected light138, either optical power is added or subtracted from the firstreflected light by the defocus system 126. As shown in FIG. 12, negativeoptical power is introduced into the first reflected light 138 byshortening the intermediate focal point 196 to a defocused lens spacing204 being less than the focal length of the second positive lens 200.The shorter length of the defocused lens spacing 204 results in adivergence of light exiting the second positive lens 200 of the defocussystem 126 as defocused 1R light 148. If positive power is introduced tothe first reflected light 138, the length of the defocused lens spacing204 is made greater than the focused lens spacing 198 resulting in aconvergence of light exiting the second positive lens 200 of the defocussystem 126.

An alternative implementation of the imaging system 100 uses a versionof the object light 132 being linearly polarized having a firstpolarization state vector 206 oriented in the x-y plane, as illustratedin FIG. 13. The collection lens 108, the beam splitter optical coating112, and the first reflector 124 act upon the object light 132, thecollected light 134, and the first reflected light 138, respectively,without affecting the orientation of the first polarization state vector206. In this implementation, the imaging system 100 uses a polarizationbeam splitter 208 having a polarization beam splitter optical coating210 being oriented in the polarization beam splitter such that the firsttransmitted light 136, with its particularly oriented first polarizationstate vector 206, passes substantially completely through thepolarization beam splitter 208 as the 2T1T light 138 due to theorientation of the first polarization state vector. After leaving thefirst reflector 124, the first reflected light 138 passes through anoptical retardation plate 212 thereby altering the first polarizationstate vector 206 to a second polarization state vector 214 beingoriented in the x-z plane, as illustrated in FIG. 13. Subsequently, thedefocused 1R light 148, having the second polarization state vector 214,is substantially completely reflected off of the polarization beamsplitter 208 of the polarization beam splitter optical coating 210 asthe 2R defocused light due to the orientation of the second polarizationstate vector. As a result of the polarization effect associated with thepolarization beam splitter 208, both the 2T1T light 138 and the 2Rdefocused light are substantially brighter compared to otherimplementations of the imaging system 100 not relying upon thepolarization effect. When compared with other implementations, opticalefficiency is approximately doubled by utilizing the polarization effectalthough there is some absorption loss associated with the opticalretardation plate 212.

An alternative implementation of the imaging system 100 uses a polarizedand un-polarized versions of the object light 132. The polarized versionof the object light 132 has a third polarization state vector 216oriented in the y-z plane and approximately 45 degrees relative to boththe y-axis and the z-axis, as illustrated in FIG. 15. The collectionlens 108 passes the object light 132 as the collected light 134 withoutaffecting polarization. A polarization beam splitter 218 having apolarization beam splitter optical coating 220 receives both polarizedand un-polarized versions of the collected light 134 and splits thecollected light into a polarized version of first transmitted light 136having the first polarization state vector 206 oriented along the y-axisplane and a polarized version of the first reflected light 138 havingthe second polarization state vector 214 oriented along the z-axis.

The first reflector 124, the and the second reflector 130 do notsubstantially alter the polarization of the first reflected light 138with the second polarization state vector. The polarization beamsplitter optical coating 210 of the polarization beam splitter 208 isoriented such that the first transmitted light 136 with the firstpolarization state vector 206 passes substantially completely throughthe polarization beam splitter optical coating of the polarization beamsplitter as 2T1T light 138 also with the first polarization state vectorand the defocused 1R light 148 with the second polarization state vector214 is substantially completely reflected off of the polarization beamsplitter optical coating of the polarization beam splitter as 2Rdefocused light also with the second polarization state vector. As aresult, an approximate doubling of optical efficiency is achieved, ascompared with other implementations, without additional expense andabsorption loss associated with use of the optical retardation plate212.

An implementation of the first detector 120, composed of pictureelements such as detector pixels arranged in detector row 222 anddetector columns 224, is illustrated in FIG. 16. Typically, such animplementation of the first detector 120 would utilized time delayintegration (TDI). Cells or other objects as the target object 102 areentrained in a fluid stream to be imaged on the first detector 120 asthey flow in a fluid flow direction 226 through the flow cell cuvette104. Sets of 2T1T focus cell images 228 in a 2T1T focus area 230 and 2Rdefocused focus cell images 232 in a 2R defocused focus area 234 areimaged on the first detector 120 along the detector columns 224. The2T1T focus area 230 and the 2R defocused focus area 234 are spatiallyseparated from one another by a suitable number of the detector pixelsto avoid image overlap. For instance, in typical implementations forimaging cells having nominally 10 micron diameters, the 2T1T objectfield of view 164 is configured to be approximately 90 microns. Given anexemplary 10 pixel separation between the 2T1T focus area 230 and the 2Rdefocused focus area 234 and with an exemplary implementation of thefirst detector 120 having 13 micron sized pixels, a satisfactory channelseparation would be 100 pixels or 1.3 mm. Furthermore, as an example, ifthe imaging system 100 were to have an overall magnification of 40×, andfocal length of 200 mm for the imaging lens 118, the optical angle ofseparation 142 between the imaged 2T1T light 144 and the imaged 2Rdefocused light 150 would be approximately 6.5 milliradians.Consequently, in this example, the mechanical angle 104 for theamplitude beam splitter 114 would be approximately 3.25 milliradians.

Another implementation of the imaging system 100, illustrated in FIGS.17 and 18, uses a spectral dispersing element 246, such as a prism ordiffraction grating, to spectrally disperse light from the amplitudebeam splitter 114, shown in FIG. 17, or from the polarization beamsplitter 208, not shown in FIG. 17, such as the 2T1T light 138 and the2R defocused light to transmit spectrally dispersed 2T1T light 248 andspectrally dispersed 2R defocused light 250. The imaging lens 118 thenreceives the spectrally dispersed 2T1T light 248 and spectrallydispersed 2R defocused light 250 to transmit imaged, spectrallydispersed 2T1T light 252 and imaged, spectrally dispersed 2R defocusedlight 254, respectively, having the 2T1T image plane 146 and the 2Rdefocused image plane 152, with respect to a common point on targetobject 102, respectively. As illustrated in FIG. 19, the 2T1T focus area230 and the 2R defocused focus area 234 have a spectrally dispersed bandof images, 2T1T focus cell dispersed image set 256 and 2R defocusedfocus cell dispersed image set 258, respectively, for each occurrence ofthe target object 102. This spectral dispersion is useful for analysisof the target object 102.

Another implementation of the imaging system 100, illustrated in FIG.20, uses an x-axis imaging system 260 and an y-axis imaging system 262to image the target object 102 bi-dimensionally from two differentorientations, which is useful, for instance, to distinguish featuresthat may otherwise overlap when viewed from a single orientation. Theparticular implementation illustrated in FIG. 20 utilizes polarizationeffects in conjunction with the optical retardation plate 212 and thepolarization beam splitter 208 and spectral dispersion effects inconjunction with the spectral dispersing element 246. However, otherimplementations can use the x-axis imaging system 260 and the y-axisimaging system 262 with or without the polarization effects and thespectral dispersion effects.

Applications of bi-dimensional implementations of the imaging system 100include analyzing multi-component objects in solution, such as cellscontaining FISH probes. Since FISH probes appear as point sources oflight within the three-dimensional nucleus of a cell, in some cases, twoor more FISH probes may appear in an overlapping relationship along theoptical axis of the imaging system. Consequently, one or more FISHprobes may obscure one or more other FISH probes to undermine attemptsat determining the quantity of FISH probes contained within a cell.Determination of FISH probe quantity within a cell has tremendousutility such as in determining genetic abnormalities, (for example,trisomy 21, otherwise known as Down's syndrome).

By positioning the optical axes of the x-axis imaging system 260 and they-axis imaging system 262 so that they are oriented with respect to oneanother by 90°, such as the optical axis of the x-axis imaging systembeing along the x-axis and the optical axis of the y-axis imaging systembeing along the y-axis, as shown in FIG. 20, it is possible toseparately resolve image spots imaged from corresponding two or moreFISH probe objects on at least one of the first detectors 120 of atleast one of the x-axis imaging system and the y-axis imaging system. Ithas been found that if two or more FISH probes overlap in regard to theimage produced on one of the first detectors 120, the two or more FISHprobes can be separately resolved in the spectrally dispersed imagesproduced on the other first detector.

This is in contrast to conventional approaches where single-orientationsystems may address problems caused by image overlap due to defocus bypanning through objects along the optical axis of the conventionalsystems to acquire multiple image planes in the object. Theseconventional approaches require significant amounts of time to collectmultiple images and cannot readily be applied to objects, such as cells,in flow. The implementation of the imaging system 100 using two imagingsub-systems, the x-axis imaging system 260 and the y-axis imaging system262, addresses image overlap problems, even while objects to be imagedare in motion, through its multi-object plane approach.

Object planes associated with an orthogonal orientation of the opticalaxis of the x-axis imaging system 260 with respect to the y-axis imagingsystem 262 are illustrated in FIG. 22. As a result of the orthogonalorientation of the optical axis of the x-axis imaging system 260 withrespect to the y-axis imaging system 262, the 2T1T object plane 156 andthe 2R defocused object plane 158 of the x-axis imaging system are alsoorthogonal with respect to the 2T1T object plane and the 2R defocusedobject plane of the y-axis imaging system.

In an alternative implementation of the imaging system 100 as abi-oriented imaging system 264, illustrated in FIG. 23, a focus feedbackerror is generated to dynamically acquire or maintain focus. Thebi-oriented imaging system 264 includes a flow cell cuvette 266, a flowcell cavity 268, an illumination light 270, a first imaging subsystem272, a first detector 274, second imaging sub-system 276, a seconddetector 278 and a processor 280. The first imaging sub-system 272receives the first collected light from a second target object 282 andtransmits first focused light 284 along a first optical axis 286 to bereceived by the first detector 274. The first focused light 284 has afirst imaging sub-system best focused conjugate image for second targetobject (first image of second target) 288 with respect to the secondtarget object 282. The first focused light 284 also has a first imagingsub-system best focused conjugate image for first target object (firstimage of first target) 290 with respect to a first target object 292also in the flow cell cavity 268. The first collected light results fromlight either being emanated from luminous versions of the second targetobject 282 or coming from an incoherent or coherent light source andbeing scattered or reflected off of the second target object. The secondimaging sub-system 276 receives the second collected light from thesecond target object 282 and transmits second focused light 294 alongsecond optical axis 296 to be focused at the second imaging subsystembest focused conjugate image for second target object (second image ofsecond target) 298. For the implementation depicted in FIG. 23, thefirst optical axis 286 and the second optical axis 296 are orthogonalwith respect to one another.

With respect to the example shown in FIG. 23, the second target object282 and the first target object 292 occupy the same position withrespect to the direction of the first optical axis 286. However, withrespect to the direction of the second optical axis 296, the firsttarget object is closer to the second imaging sub-system 276 than is thesecond target object. As shown in FIG. 23, a first lateral shift 300exists between the first image of second target 288 and the first imageof first target 290 along the surface of the first detector 274 sincethe second target object 282 and the first target object 292 occupy thesame position with respect to the first optical axis 286 and not withrespect to the orthogonal second optical axis 296. As shown in FIG. 23,the second detector 278 is located with respect to the best focusposition for the first target object 292. Since the second target object282 is farther away than the first target object 292 from the secondimaging sub-system 276, the second image of second target 298 is locateda focus shift for second imaging sub-system image 302 from the seconddetector 278.

Due to the orientation between the first imaging sub-system 272 and thesecond imaging sub-system 276, the focus shift for second imagingsub-system image 302 is proportional to the first lateral shift 300. Insome implementations, the processor 280 is communicatively linked bycommunication links 503 to the first detector 274 and/or the seconddetector 278 to determine lateral displacements such as the firstlateral shift 300. The processor 280 can further be communicativelylinked by the communication links 503 to the first detector 274, seconddetector 278, the first imaging sub-system 272, and/or the secondimaging sub-system 276 to either adjust the position of the firstdetector or the second detector, or adjust optical characteristics ofthe first imaging sub-system or the second imaging sub-system based upondetermined displacements to correct for focus shifts such as the focusshift for second imaging sub-system image 302. For instance, asillustrated in FIG. 23, the processor 280 could determine that the firstlateral shift 300 occurred as the first focused light 284 moved fromfirst image of first target 290 to the first image of second target 288as the flow cell cavity 268 first contained the first target object 292and then contained the second target object 282. As a consequence ofthis determination, the processor 280 would instruct the second imagingsub-system 276 to move the second image of second target 298 to thesecond detector 278 based upon the first lateral shift 300.Alternatively, the processor 280 would instruct the second detector 278to move to the current position of the second image of second target 298as shown in FIG. 23.

The relationship between lateral shifts, such as the first lateral shift300, and focus shifts, such as the focus shift for second imagingsub-system image 302, is further elaborated by use of FIG. 24 showing arepresentative example of the first detector 274 having a plurality of afirst detector picture element 304, each being approximately 10 micronsin size, in this representative example, arranged in rows and columns.The first image of first target 290 of the first target object 292 isshown as an exemplary cell having a cytoplasm and cell nucleus. Sincethe first target object 292 is in focus at the second image of secondtarget 298, the lateral position along the x-axis of the centroid offirst image of second target 308 of its first image of first target 290defines the location of the ideal focal plane for a orthogonal axis 306along the y-axis on the first detector 274. The first image of secondtarget 288 of the second target object 282 is also shown as a cellhaving a cytoplasm and cell nucleus. Since the second target object 282is not positioned at the ideal focal plane for the second detector 278,the second target object is imaged off-axis on the first detector 274and a centroid of first image of first target 310 of its first image ofsecond target 288 exhibits the first lateral shift 300 in position fromthe on-axis of the first image of first target 290 of the first targetobject 292. The amount of lateral shift between images at the firstdetector 274 is determined by the separation of objects, such as thefirst target object 292 and the second target object 282, and thelateral magnification of the first imaging sub-system 272. The amount ofdefocus between images at the second detector 278 is determined by theseparation of the objects and the magnification along the second opticalaxis 296 or the longitudinal magnification of the second imagingsub-system 276. It is to be noted that the longitudinal magnification ofthe optical system, in these examples, is equal to the square of thelateral magnification.

In a typical implementation, magnification of optical systems such asthe first imaging sub-system 272 in the second imaging sub-system 276 is10×, with a pixel size on the detectors, such as the first detector 274and the second detector 278, being 10 microns. In FIG. 24, a five pixelor 50 micron positive lateral shift along the x-axis on the firstdetector 274 for the centroid of first image of first target 310 isshown. In this representative example, given a 10×magnification, a 50micron positive lateral shift along the x-axis translates into a fivemicron shift along the optic axis, such as the second optical axis 296,away from the second imaging sub-system 276. In order to correct focus,the second detector 278 should be moved approximately 500 microns (fivemicron error×lateral magnification×lateral magnification) toward thesecond imaging sub-system 276. In these implementations, centroids, suchas the centroid of first image of second target 308 and the centroid offirst image of first target 310, are calculated using conventionalmethods. Some implementations keep a running average of multiple cellcentroid locations to normalize any inconsistencies in cell shape beforeinstructing an electromechanical system associated with either thedetectors, such as the first detector 274 and the second detector 278,or optical subsystems, such as the first imaging sub-system 272 and thesecond imaging sub-system 276, for correction of focus error.

In general, information from each of the imaging sub-systems, such asthe first imaging sub-system 272 in the second imaging sub-system 276,may be used to correct focus of one another. The target objects, such asthe second target object 282, the first target object 292, and othertarget objects including other types of cells, do not need to lie alongone of the optical axes of the imaging sub-systems, such as the firstoptical axis 286 and the second optical axis 296 in order to determinecentroids of the target objects and to ascertain lateral shift.Implementations are used with magnification at various levels as long ascorresponding lateral displacements are properly translated into focuserror and subsequently proper correction is implemented. Many sorts ofelements conventionally known can be translated in order to correct forfocus error; therefore, the representative examples related to theseimplementations are not meant to be limiting. In other implementations,other types of detectors are used such that images of the target objectsare not created, but rather only centroids are computed that areindicative of the position of the one or more target objects in the flowcell cavity 268.

As in the un-polarized implementations of the imaging system 100, it isimportant to control the amount of light in each beam path in order toresult in images of approximately the same intensity level at thedetector. In addition to the methods previously discussed for lightcontrol, in the polarized implementations, the light intensity in thedefocus optical path 138 can also be controlled by the angularorientation of the optical retardation plate 212. As the opticalretardation plate is rotated the plane of linear polarization alsorotates. This results in the second polarization state vector 214 at thepolarization beam splitter 208 to be rotated with respect to the planeof incidence so that polarization beamsplitter optical coating 116splits the incident light into its vector component s- andp-polarization states. Since the p-polarized light is transmittedthrough the polarization beamsplitter optical coating 116 while thes-polarized light is reflected, the 2R defocused light light 168 isreduced in intensity. The effective beamsplitter ratio at thepolarization beam splitter 208 can therefore be varied in this manner.An alternative to the use of neutral density filters in the polarizedembodiment is the use of a linear polarizer as a variable transmittancefilter. When placed in the linear polarized first transmitted light 136or the first reflected light 138, the transmittance of the light throughthe polarizer will vary with the orientation of the polarizer axis.

An active autofocus system 700 is illustrated in FIG. 25 to receive froman optical system 702 such as an implementation of the imaging system100, light 704 by a camera 708. The host computer 720 runs an auto focussystem software having a method such as described below. Object light132 is collected from the target object 108 and image by the opticalsystem 702 onto the camera 708. Light 704 from the optical system isbrought to a focus at the camera 708 with the precise focal positionunder control involving a frame grabber 712, an image processor 716, ahost computer 720, a motor driver 724, and a motorized 728. Themotorized stage 728 may be configured to move the entire optical system700 or any number of optical components of the optical system, such asthe camera 708. Alternatively, the motorized stage 728 could beconfigured to move the target object 102. The host computer 720 controlsimage acquisition by the camera 708. The frame grabber 712 executesmethods for image processing 716, which result in instructions based onone or more autofocus error signals being sent to the motor driver 724to move the motorized stage 728 the appropriate magnitude and directionso as to maintain objects in focus at the camera.

A method 800 for maintaining objects in focus using the active autofocussystem 700 is shown in FIG. 26. The method 800 works in conjunction withthe optical system 702, such as the imaging system 100 wherein in theimaging system produces imagery such as described with respect to adetector, such as the first detector 120, shown in FIG. 16 having twofocus areas, such as the 2T1T focus area 224 and the 2R defocused area232. Imagery is used by the method 800 to produce a focus error signalused to control the position of an adjustable optical component of theautofocus system 700, as described above, to maintain the imagery infocus. The method 800 begins with sample flow being initiated (step 808)and an image being acquired (step 812).

Segmentation processes are used to identify objects of interest (e.g.cells) in the two focus areas (step 816). For these segmented objects,their frequency content is analyzed for each image column (focal plane)(step 820) and compared with each other (step 824) to determine whetherthe frequency content is balanced, e.g. when the system is in focus. Ifthe frequency content is balanced (YES branch of decision step 828), thesystem is in focus and no focus correction is required, so the method800 determines whether additional samples remain and if not (NOT branchof decision step 848) ends. Otherwise (YES branch of decision step 848)goes back to step 812. If the frequencies are not balanced (NO branch ofdecision step 828), an focus error signal is determined (step 836) (e.g.from the ratio of frequency content) and the required focal shiftmagnitude and direction is determined (step 840) by reference to adatabase of stored correction factors or a look-up table. The refocusingoptics are then adjusted (step 844) in the proper direction by therequired amount and step 848 is executed as described above.

An alternative implementation 900 of the imaging system 100 isillustrated in FIG. 27 wherein one reflector 904 is used to reflectlight in the defocus optical path 122. In this exemplary illustration ofthe alternative implementation, converging light 902 is received by theamplitude beam splitter 110 and is partially reflected and partiallytransmitted. The portion of the converging light 902 that is partiallyreflected is first defocused through the defocus system 126 and thenreflected by a reflector 904 on to the amplitude beam splitter 114 to bepartially reflected as defocused light 908. The portion of theconverging light 902 that is partially transmitted by the amplitude beamsplitter 110 is also partially transmitted by the amplitude beamsplitter 114 as unaltered light 906.

And exemplary implementation of the imaging system is illustrated inFIG. 28 showing the defocus system 126 positioned in the transmissionpath of the amplitude beam splitter 110. As a result, unaltered light920 and defocused light 922 have reversed positions compared to otherimplementations described above. In other implementations using otheraspects described above, including but not limited to polarizationaspects, dispersion aspects, bi-orientation aspects, and aspectsdirected to other multiple detector configurations, the defocus system126 is also position in a transmission path rather than a reflectedpath.

The numerical aperture, NA, of a microscope objective lens is given byn*sin θ where n is the index of refraction of the medium in which theobject lies and θ is the half angle of the cone of collected light. Thedepth of focus of an optical system is the distance through which adetector can be moved along the optical axis forward and backward fromfocus before the image appears to be out of focus. For adiffraction-limited lens such as a well-corrected microscope objective,Rayleigh's criterion for tolerable defocus allows for λ/4 wave ofwavefront error where λ is the wavelength of the image forming light.This translates to an allowable depth of focus at the image ofD′=λ/(NA′)²where NA′ is the numerical aperture on the image side of the objective.For a system with lateral magnification m, NA′=NA/m andD′=m ^(2*)λ/(NA)²where NA is the numerical aperture on the object side of the objective.The depth of field, D, is related to the depth of focus by thelongitudinal magnification of the system, m², so that D=D′/m² orD=λ/(NA)²For an oil immersion type objective the index of refraction of the oilmust be accounted for and the depth of field is n times larger than theabove.

High numeric aperture microscope objectives used with some of theimplementations of the imaging system 100 are readily availablecommercially with NA values ranging from 0.5 to 1.4. For visible lightimaging, assuming a center wavelength of λ=0.55 microns, these NA valuestranslate to tolerable depths of field from as little as 0.4 microns to4.0 microns. Tolerances for allowable depth of focus other thanRayleigh's criterion may result in an expansion or reduction of thisrange. For example, a decrease in the modulation transfer function at aparticular spatial frequency might be the acceptance criterion forimplementation of the imaging system 100.

In some implementations of the imaging system 100 for biological cellimaging in flow, collection lens are microscope objectives of 40×magnification with 0.9 NA and the imaging lens has a focal length of 200mm. Cell objects are nominally 10 microns in diameter and the imagingfield of view orthogonal to the flow axis is set to be 30 microns.Detector pixel size is approximately 13 microns. Consequently, thedesired lateral separation between unaltered and defocused focal planeimages at the detector is 100 pixels or 1.3 mm. The lateral separationat the detector is given by f*tan φ, where f is the focal length of theimaging lens and φ is the optical angle of separation. For the 200 mmfocal length lens the angle of separation is 6.5 milliradians to achievethe 1.3 mm lateral separation. Note that this translates to a mechanicalangle of 3.25 milliradians for the beam combiner element, since uponreflection the optical angle is twice the mechanical angle of thereflective surface. The depth of field for the 0.9NA objective is 1.03microns and the required optical power introduced into the defocusedoptical path is ±0.04 diopter, corresponding to a defocus lens focallength of ±25 meters. This optical power results in a separation of theunaltered and defocused object planes by 1 micron, to nearly double thedepth of field of the system.

Numerous implementations of the imaging system 100 can be accomplishedwith a variety of components. In the biological application objects arecells of typically 5 to 20 microns in diameter. In otherimplementations, microscopic objects of interest may have a size rangeof 1 to 50 microns. High NA microscope objectives are commerciallyavailable from 10×/0.5NA to 100×/1.4NA with optical designs optimizedfor use with imaging lens focal lengths of 165 to 200 mm. Typical CCDdetector pixel sizes range from 5 to 25 microns. Optical systemsemploying these components in various embodiments may require opticalpower in the defocused optical path to range from ±0.01 to ±0.1 diopter.Angular separation between the unaltered and defocused optical paths mayrange from as little as 0.1 degree to 10 degrees. However, those skilledin the art will appreciate that other optical system applications withdifferent imaging requirements can be constructed with custom designedcomponents that may extend these typical parameter ranges.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. For imaging a target object, an imaging system comprising: a firstbeam splitter configured to substantially transmit part of receivedlight as first transmitted light and to substantially reflect part ofreceived light as first reflected light; a defocus system configured tomodify optical power of substantially one of the following: the firsttransmitted light and the first reflected light, and to transmit thesame as first transmitted defocused light; a reflector configured toreflect one of the following: the first reflected light and the firsttransmitted defocused light; a second beam splitter configured tosubstantially transmit part of one of the following: the firsttransmitted light as second transmitted light and the first transmitteddefocused light as second transmitted defocused light and configured tosubstantially reflect part of one of the following: the firsttransmitted defocused light as second reflected defocused light and thefirst reflected light as second reflected light; and an imagingsub-system configured to focus one of the following pairs of light: thepair of the second transmitted light as imaged unaltered light and thesecond reflected defocused light, as imaged defocused light and the pairof the second transmitted defocused light as imaged defocused light andthe second reflected light as imaged unaltered light and to focus theimaged defocused light with respect to an imaged defocused image planeand the imaged unaltered light with respect to an imaged unaltered imageplane separated from the imaged defocused image plane, the second beamsplitter oriented according to a mechanical angle such that the imagedunaltered light and the imaged defocused light have an angularseparation other than zero.
 2. The system of claim 1 wherein the secondbeam splitter is further configured to transmit part of the firstreflected light and reflect part of the first transmitted light.
 3. Thesystem of claim 1 wherein the defocus system is a negative lens.
 4. Thesystem of claim 1, further comprising a collection system wherein thecollection system is a lens.
 5. The system of claim 1, furthercomprising an optical retardation plate and wherein the second beamsplitter is a polarization beam splitter having a polarization beamsplitter optical coating.
 6. The system of claim 1 wherein the imagingsub-system is an imaging lens.
 7. The system of claim 1 wherein thefirst beam splitter and the second beam splitter are polarization beamsplitters having polarization beam splitter optical coatings.
 8. Thesystem of claim 1, further including a first detector positioned toreceive the imaged defocused light with respect to the imaged defocusedimage plane and the imaged unaltered light with respect to the imagedunaltered image plane.
 9. The system of claim 8, further comprising asecond imaging sub-system and a second detector.
 10. The system of claim8 wherein the detector has first and second focus areas.
 11. The systemof claim 1, further comprising a spectral dispersing element configuredto transmit light to the imaging sub-system.
 12. The system of claim 1,further comprising: a secondly-oriented first beam splitter configuredto substantially transmit part of the collected light as firsttransmitted light and to substantially reflect part of the collectedlight as first reflected light; a secondly-oriented defocus systemconfigured to modify optical power of substantially one of thefollowing: the first transmitted light and the first reflected light totransmit as defocused light; a secondly-oriented reflector configured toreflect one of the following: the first reflected light and thedefocused light; a secondly-oriented second beam splitter configured tosubstantially transmit part of one of the following: the firsttransmitted light as second transmitted first transmitted light and thedefocused light as second transmitted defocused light and configured tosubstantially reflect one of the following: the defocused light assecond reflected defocused light and the first reflected light as secondreflected first reflected light; a secondly-oriented imaging sub-systemconfigured to focus one of the following pairs of light: the pair of thesecond transmitted first transmitted light as imaged unaltered light andthe second reflected defocused light as imaged defocused light and thepair of the second transmitted defocused light as imaged defocused lightand the second reflected first reflected light as imaged unaltered lightand to focus the imaged defocused light with respect to a imageddefocused image plane and the imaged unaltered light with respect to animaged unaltered plane being separated from the imaged defocused plane;and a secondly-oriented first detector positioned to receive the imageddefocused light and the imaged unaltered light, the secondly-orientedsecond beam splitter oriented according to a mechanical angle such theunaltered light and the defocused light have an angular separation. 13.For imaging a target object, an imaging system comprising: a first beamsplitter configured to substantially transmit part of collected light asfirst transmitted light and to substantially reflect part of collectedlight as first reflected light; a defocus system configured to modifyoptical power of substantially one of the following: the firsttransmitted light and the first reflected light to transmit as defocusedlight; a reflector configured to reflect one of the following: the firstreflected light and the defocused light; a second beam splitterconfigured to substantially transmit part of one of the following: thefirst transmitted light as unaltered light and the defocused light asdefocused light and configured to substantially reflect part one of thefollowing: the defocused light as defocused light and the firstreflected light as unaltered light; and a first detector positioned toreceive the defocused light and the unaltered light with respect to afirst object plane and the defocused light with respect to a secondobject plane, the defocus system configured to modify optical power suchthat the first depth of focus overlaps the second depth of focus, thesecond beam splitter oriented according to a mechanical angle such theunaltered light and the defocused light have an angular separation. 14.For imaging a target object, an imaging system comprising: a first beamsplitter configured to substantially transmit part of collected light asfirst transmitted light and to substantially reflect part of collectedlight as first reflected light; a defocus system configured to modifyoptical power of substantially one of the following: the firsttransmitted light and the first reflected light to transmit as defocusedlight; a reflector configured to reflect one of the following: the firstreflected light and the defocused light; and a second beam splitterconfigured to substantially transmit part of one of the following: thefirst transmitted light as unaltered light and the defocused light asdefocused light and configured to substantially reflect part one of thefollowing: the defocused light as defocused light and the firstreflected light as unaltered light, the second beam splitter orientedaccording to a mechanical angle such the unaltered light and thedefocused light have an angular separation.
 15. For imaging a targetobject, a method comprising: substantially transmitting part of receivedlight as first transmitted light and to substantially reflect part ofreceived light as first reflected light; modifying optical power ofsubstantially one of the following: the first transmitted light and thefirst reflected light, and to transmit the same as first transmitteddefocused light; reflecting one of the following: the first reflectedlight and the first transmitted defocused light; transmitting part ofone of the following: the first transmitted light as second transmittedlight and the first transmitted defocused light as second transmitteddefocused light and configured to substantially reflect part of one ofthe following: the first transmitted defocused light as second reflecteddefocused light and the first reflected light as second reflected light;and focusing one of the following pairs of light: the pair of the secondtransmitted light as imaged unaltered light and the second reflecteddefocused light, as imaged defocused light and the pair of the secondtransmitted defocused light as imaged defocused light and the secondreflected light as imaged unaltered light and to focus the imageddefocused light with respect to an imaged defocused image plane and theimaged unaltered light with respect to an imaged unaltered image planeseparated from the imaged defocused image plane, the second beamsplitter oriented according to a mechanical angle such the imagedunaltered light and the imaged defocused light have an angularseparation other than zero.